Metabolomics Study Of Phytosterol Esters On Prevention Of NAFLD Using UPLC Q-TOF/MS

The lifestyle of eating high-fat and high-calorie diet in today's society has led to an increase in the incidence of nonalcoholic fatty liver disease(NAFLD).NAFLD can be regarded as the pathological state of metabolic syndrome in the liver.If not timely intervention,hepatic inflammatory lesions,fibrosis,and cirrhosis of the liver will occur further.Therefore,the prevention of NAFLD has received extensive attention.Phytosterol esters(PSE),an esterified product from Phytosterols(PS),have good solubility characteristics.Our previous study found that PSE can significantly reduce the degree of steatosis in the liver of NAFLD rats.In this study,a NAFLD rat model was induced by high-fat diet,and low(0.05 g/100 g BW/d),high dose(0.10 g/100 g BW/d)PSE fortified milk was administered by gavage.The total fat content of the liver was tested.The effect of PSE on the small molecule metabolites in the liver,serum and feces of NAFLD rats was analyzed by Ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS~E)technique.The Progenesis QI v2.3 software was used to analyze unsupervised and supervised model.UNIFI data was used to analyze the retention time of different metabolites in the platform,along with the structure and lysis rules of different metabolites in each group.The specific metabolic pathway was determined by matching the metabolites with the Metabo Analyst database.The differential metabolites obtained were correlated with the degree of hepatic steatosis by SPSS,and the metabolic molecular mechanism by which PSE plays a preventive role against NAFLD was studied.The research results are as follows:(1)The study of small molecule metabolites in the liver found 20different metabolites including phosphatidic acid(16:0/20:2(11Z,14Z),20:1(11Z)/0:0),phosphatidylcholine(16:0)/18:1(11E),18:1(6Z)/0:0,18:1(9Z)/18:0,20:3(8Z,11Z,14Z)/0:0),phosphatidylethanolamine(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z),22:6(4Z,7Z,10Z,13Z,16Z,19Z)/17:1(9Z)),phosphatidylglycerol(17:2(9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)),lysophospholipid(18:1(9Z),20:3(5Z),20Z,11Z)),20:3(8Z,11Z,14Z)),etc.PSE intervention can reduce the ionic strength of phosphatidic acid,phosphatidylcholine,phosphatidylcholine,etc.(2)The study of serum small molecule metabolites found a total of 27different metabolites,including diglyceride(14:0/22:2(13Z,16Z)/0:0,15:0/18:3(6Z,9Z,12Z)/0:0,16:0/20:3(8Z,11Z,14Z)/0:0,18:1(11Z)/18:1(11Z)/0:0),lysophospholipid(15:0,16):1(9Z),20:3(5Z,8Z,11Z),20:3(8Z,11Z,14Z)),phosphatidylcholine(16:0/20:3(5Z,8Z,11Z),16:0/P-18:0,18:1(11Z)/20:1(11Z)),phosphatidylethanolamine(16:0/0:0,18:3(6Z,9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z),20:3(8Z,11Z,14Z)/0:0),etc.PSE intervention down-regulated the ionic strength of serum diglyceride,lysolecithin,phosphatidylcholine,phosphatidylethanolamine,phosphatidic acid,phosphatidylinositol,etc.,and up-regulated the ionic strength of sphingomyelin,ceramide,and glycosphingolipid.(3)Screening of fecal small molecule metabolites revealed 13differential metabolites,including 3?,12?,15?-trihydroxy-5?-chol-8(14)-ene-24-acid,11-deoxy-11-methylene-PGD 2,iso(4E,15-methyl-D16:1)sphingosine,phosphatidylglycerol,phosphatidylinositol,(R)-10-hydrox ystearic acid,etc.Besides,The ionic strength of bile acids and most lipids was significantly reduced after PSE intervention.(4)Correlation analysis between differential metabolites and degree of steatosis revealed that substances,including phosphatidylcholine,phosphatidic acid in the liver and serum,and phosphatidylethanolamine in the liver,lysophospholipid(18:1(9Z))in serum and phosphatidylglycerol(22:4(7Z,10Z,13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z))in feces,showed positive correlation with steatosis.Conclusion:Phosphatidylcholine(15:0/18:1(11Z),16:0/18:1(11E),18:1(9Z)/18:0,16:0/16:1(9Z),20:3(8Z,11Z,14Z)/0:0,19:3(10Z,13Z,16Z)/0:0,17:1(10Z)/0:0,18:1(6Z)/0:0),phosphatidic acid(16:0/20:2(11Z,14Z)),phosphatidylethanolamine(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z),16Z,19Z))in the liver,phosphatidylcholine(16:0/20:3(5Z,8Z,11Z),20:3(8Z,11Z,14Z)/18:0,17:1(10Z)/0:0),phosphatidic acid(22:4(7Z,10Z,13Z,16Z)/0:0),lysophospholipid(18:1(9Z))in serum and phosphatidylglycerol(22:4(7Z,10Z,13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z))in feces can be used as an auxiliary diagnostic indicator of NAFLD.PSE can prevent NAFLD by regulating the metabolism of glycerophospholipid induced by high-fat diet and the disorder of ether lipid metabolism pathway.